The Sho gene from Petunia hybrida encodes an enzyme responsible for the synthesis of plant cytokinins. The 3' region of the Sho gene contains a promoter in the opposite orientation that produces a partially overlapping antisense transcript. Although Sho expression varies significantly in individual cell types, the sense and antisense transcript levels maintain a stable ratio in most tissue types. In reporter lines for the antisense promoter, we observed a change in antisense promoter activity in newly formed tissue that had been induced by prolonged culture on cytokinins or following decapitation. We interpret these data as a reflection of tissue-specific threshold levels for activation of the antisense transcript. In all tissue types tested, we detect a pool of antisense RNA of approximately 35 nt, which derives from the region where Sho sense and antisense transcripts overlap. We detect a second pool of putative dsRNA breakdown products of approximately 24 nt in all tissues tested, except roots, which are the main source of cytokinin synthesis. Our data suggest that antisense transcription can be activated in a tissue-specific manner to adjust local cytokinin synthesis via degradation of Sho dsRNA. We therefore propose that, in addition to cytokinin transport and inactivation, regulation of local cytokinin synthesis via antisense transcription represents yet another device for the complex control of local cytokinin levels in plants.